4.3 DNA Sequencing
Determination of precise sequence of nucleotide in sample DNA
Maxam-Gilbert base destruction method
bases DNA selectively destroyed
not use anymore (reagent toxic&dangerous)
used purified DNA
labelling 5' end (add gamma 32P ATP)
break into G, A+G, C, C+T
Sanger Method
by Frederick Sanger in 1980
DNA sequence as single strand (by heat)
template DNA supplied w mix of all dNTPs, primer sequence complement to 3' end
diff tube of all four dideoxynucleotide present in limit quantities and label w tag that fluorescence
chain elongation cut off after DNA polymerase insert ddNTP instead of dNTPs
chain termination, primer has defined length of 32P at 5' end and hybridize at 3' end of template strand
generate a series of fragment then use to read the nucleotide sequence
running the product in each tube-on a gel allow to determine each chain terminating ddNTPs was incorporated
stop synthesis selectively at any four ddNTPs
produce a population of molecule
separated by length from the longest to shortest
diff of one nucleotide is enough to separate from other
each ddNTPs light a diff color when illuminated by a laser beam